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• W2058258296 abstract "A putative oncogene bcl-3 was originally identified and cloned at the breakpoint in the recurring chromosome translocation t(14;19) found in some cases of B cell chronic lymphocytic leukemia. Studies of bcl-3-deficient mice demonstrated a critical role for bcl-3 in the development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. However, the molecular mechanism that underlies B cell leukemogenesis and the knockout mouse phenotype remains unclear. Here we have identified and characterized BCL-3-binding protein (B3BP) as a protein interacting specifically with the bcl-3 gene product (BCL-3) by a yeast two-hybrid screen. We found that B3BP associates with not only BCL-3 but also p300/CBP histone acetyltransferases. The N-terminal region of B3BP that contains the ATP-binding site is important for the interaction with BCL-3 and p300/CBP. Homology searches indicate that the ATP-binding region of B3BP, which contains a typical Walker-type ATP-binding P-loop, most resembles that of 2′,3′-cyclic nucleotide 3′-phosphodiesterase of mammals and polynucleotide kinase of T4 bacteriophage. In fact B3BP shows intrinsic ATP binding and hydrolyzing activity. Furthermore, we demonstrated that B3BP is a 5′-polynucleotide kinase. We also found a small MutS-related domain, which is thought to be involved in the DNA repair or recombination reaction, in the C-terminal region of B3BP, and it shows nicking endonuclease activity. These observations might help to gain new insights into the function of BCL-3 and p300/CBP, especially the coupling of transcription with repair or recombination. A putative oncogene bcl-3 was originally identified and cloned at the breakpoint in the recurring chromosome translocation t(14;19) found in some cases of B cell chronic lymphocytic leukemia. Studies of bcl-3-deficient mice demonstrated a critical role for bcl-3 in the development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. However, the molecular mechanism that underlies B cell leukemogenesis and the knockout mouse phenotype remains unclear. Here we have identified and characterized BCL-3-binding protein (B3BP) as a protein interacting specifically with the bcl-3 gene product (BCL-3) by a yeast two-hybrid screen. We found that B3BP associates with not only BCL-3 but also p300/CBP histone acetyltransferases. The N-terminal region of B3BP that contains the ATP-binding site is important for the interaction with BCL-3 and p300/CBP. Homology searches indicate that the ATP-binding region of B3BP, which contains a typical Walker-type ATP-binding P-loop, most resembles that of 2′,3′-cyclic nucleotide 3′-phosphodiesterase of mammals and polynucleotide kinase of T4 bacteriophage. In fact B3BP shows intrinsic ATP binding and hydrolyzing activity. Furthermore, we demonstrated that B3BP is a 5′-polynucleotide kinase. We also found a small MutS-related domain, which is thought to be involved in the DNA repair or recombination reaction, in the C-terminal region of B3BP, and it shows nicking endonuclease activity. These observations might help to gain new insights into the function of BCL-3 and p300/CBP, especially the coupling of transcription with repair or recombination. bcl-3 was originally identified at the breakpoint in the t(14; 19) chromosome translocation in some cases of chronic B cell lymphocytic leukemia and shown to be up-regulated transcriptionally in peripheral blood lymphocytes from patients with the corresponding translocation. Therefore, its involvement in the pathogenesis of chronic B cell lymphocytic leukemia has been strongly suggested (1Ohno H. Takimoto G. McKeithan T.W. Cell. 1990; 60: 991-997Abstract Full Text PDF PubMed Scopus (361) Google Scholar). It was also reported that bcl-3 gene expression is induced by many cell growth- or survival-promoting factors in lymphocytic cell lines (2Rebollo A. Dumoutier L. Renauld J.C. Zaballos A. Ayllon V. Martinez A.C. Mol. Cell. Biol. 2000; 20: 3407-3416Crossref PubMed Scopus (95) Google Scholar, 3Richard M. Louahed J. Demoulin J.B. Renauld J.C. Blood. 1999; 93: 4318-4327Crossref PubMed Google Scholar, 4Zhang M.Y. Harhaj E.W. Bell L. Sun S.C. Miller B.A. Blood. 1998; 92: 1225-1234Crossref PubMed Google Scholar), suggesting the close involvement of bcl-3 in cell proliferation and survival. Subsequent studies showing a correlation of bcl-3 induction with mouse skin carcinogenesis (5Budunova I.V. Perez P. Vaden V.R. Spiegelman V.S. Slaga T.J. Jorcano J.L. Oncogene. 1999; 18: 7423-7431Crossref PubMed Scopus (112) Google Scholar), human breast cancer (6Cogswell P.C. Guttridge D.C. Funkhouser W.K. Baldwin Jr., A.S. Oncogene. 2000; 19: 1123-1131Crossref PubMed Scopus (377) Google Scholar), and hepatocyte proliferation (7Feng X. Jiang Y. Meltzer P. Yen P.M. J. Biol. Chem. 2001; 276: 15066-15072Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) have indicated its involvement in carcinogenesis and the growth of cells other than B lymphocytes. In fact, ectopic expression of bcl-3 blocked interleukin-4 deprivation-induced apoptosis of a T cell line in vitro and also increased the survival rate of activated T cells in vivo (2Rebollo A. Dumoutier L. Renauld J.C. Zaballos A. Ayllon V. Martinez A.C. Mol. Cell. Biol. 2000; 20: 3407-3416Crossref PubMed Scopus (95) Google Scholar, 8Hildeman D.A. Zhu Y. Mitchell T.C. Kappler J. Marrack P. Curr. Opin. Immunol. 2002; 14: 354-359Crossref PubMed Scopus (214) Google Scholar, 9Mitchell T.C. Hildeman D. Kedl R.M. Teague T.K. Schaefer B.C. White J. Zhu Y. Kappler J. Marrack P. Nat. Immunol. 2001; 2: 397-402Crossref PubMed Google Scholar). Transgenic mice in which bcl-3 is expressed under the control of an Ig heavy chain enhancer showed an expansion of the B cell population (10Ong S.T. Hackbarth M.L. Degenstein L.C. Baunoch D.A. Anastasi J. McKeithan T.W. Oncogene. 1998; 16: 2333-2343Crossref PubMed Scopus (62) Google Scholar). Nevertheless, there is no experimental evidence of a role for bcl-3 in cell transformation. Extensive biochemical study has revealed some of the molecular functions of bcl-3. First, amino acid sequence alignment showed that BCL-3 contains seven repeats of an ankyrin-like unit and belongs to the IκB family of proteins, which modulate the DNA binding activity and subcellular localization of the transcription factor NF-κB (11Nolan G.P. Baltimore D. Curr. Opin. Genet. Dev. 1992; 2: 211-220Crossref PubMed Scopus (154) Google Scholar). Subsequently, it was demonstrated that BCL-3 physically associates with the p50 and p50B homodimers of NFKB1 and NFKB2, respectively, and confers transcriptional activation to the otherwise inert complex; hence it functions as a transcriptional co-activator (12Fujita T. Nolan G.P. Liou H.C. Scott M.L. Baltimore D. Genes Dev. 1993; 7: 1354-1363Crossref PubMed Scopus (346) Google Scholar, 13Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (421) Google Scholar). We have previously demonstrated that BCL-3 induces the nuclear translocation of the p50 homodimer generated via reorganization from cytoplasmic p50/p105 (14Watanabe N. Iwamura T. Shinoda T. Fujita T. EMBO J. 1997; 16: 3609-3620Crossref PubMed Scopus (97) Google Scholar). This BCL-3-induced p50 homodimer formation has been observed in vivo; that is, ectopic expression of BCL-3 in thymocytes induced the formation of the p50 homodimer (15Caamano J.H. Perez P. Lira S.A. Bravo R. Mol. Cell. Biol. 1996; 16: 1342-1348Crossref PubMed Scopus (88) Google Scholar), and stimulation of cultured T cells with interleukin-9 leads to the induction of BCL-3 expression, which is followed by p50 homodimer generation (3Richard M. Louahed J. Demoulin J.B. Renauld J.C. Blood. 1999; 93: 4318-4327Crossref PubMed Google Scholar). Moreover, p50 homodimer has been implicated in cell survival or the inhibition of apoptosis (3Richard M. Louahed J. Demoulin J.B. Renauld J.C. Blood. 1999; 93: 4318-4327Crossref PubMed Google Scholar, 16Kurland J.F. Kodym R. Story M.D. Spurgers K.B. McDonnell T.J. Meyn R.E. J. Biol. Chem. 2001; 276: 45380-45386Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Subsequently, it has been shown that BCL-3 interacts with general transcription factors (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), other transcriptional co-activators (18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19Dechend R. Hirano F. Lehmann K. Heissmeyer V. Ansieau S. Wulczyn F.G. Scheidereit C. Leutz A. Oncogene. 1999; 18: 3316-3323Crossref PubMed Scopus (263) Google Scholar), and also other DNA-binding factors (17Na S.Y. Choi H.S. Kim J.W. Na D.S. Lee J.W. J. Biol. Chem. 1998; 273: 30933-30938Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Na S.Y. Choi J.E. Kim H.J. Jhun B.H. Lee Y.C. Lee J.W. J. Biol. Chem. 1999; 274: 28491-28496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), all of which indicates a general role for BCL-3 in the transcriptional activation. Recently, it has also been demonstrated that a putative BCL-3 ortholog of Caenorhabditis elegans interacts with MRT-2 cell cycle checkpoint protein and indeed is involved in the DNA damage response by protein-protein interaction screening combined with a large scale phenotypic analysis (20Boulton S.J. Gartner A. Reboul J. Vaglio P. Dyson N. Hill D.E. Vidal M. Science. 2002; 295: 127-131Crossref PubMed Scopus (240) Google Scholar). Above all, knockout mouse studies gave rise to important information on the biological role of BCL-3 (21Schwarz E.M. Krimpenfort P. Berns A. Verma I.M. Genes Dev. 1997; 11: 187-197Crossref PubMed Scopus (140) Google Scholar, 22Franzoso G. Carlson L. Scharton-Kersten T. Shores E.W. Epstein S. Grinberg A. Tran T. Shacter E. Leonardi A. Anver M. Love P. Sher A. Siebenlist U. Immunity. 1997; 6: 479-490Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 23Paxian S. Merkle H. Riemann M. Wilda M. Adler G. Hameister H. Liptay S. Pfeffer K. Schmid R.M. Gastroenterology. 2002; 122: 1853-1868Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The BCL-3-deficient mouse was susceptible to certain kinds of pathogens. Antigen-specific antibody production was severely impaired because germinal center formation in secondary lymphoid organs was markedly inhibited. Such a phenotype was at least to some extent similar to that of NFKB1 and NFKB2 knockout mice (23Paxian S. Merkle H. Riemann M. Wilda M. Adler G. Hameister H. Liptay S. Pfeffer K. Schmid R.M. Gastroenterology. 2002; 122: 1853-1868Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 24Sha W.C. Liou H.C. Tuomanen E.I. Baltimore D. Cell. 1995; 80: 321-330Abstract Full Text PDF PubMed Scopus (1051) Google Scholar, 25Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Crossref PubMed Scopus (363) Google Scholar, 26Poljak L. Carlson L. Cunningham K. Kosco-Vilbois M.H. Siebenlist U. J. Immunol. 1999; 163: 6581-6588PubMed Google Scholar), suggesting physiological significance of the interaction of BCL-3 with these proteins. During the development of germinal centers, B cell-specific genetic recombination of the Ig gene, class switch recombination, and somatic hypermutation proceed to produce a large amount of Ig that has a much higher affinity for the antigen. One hypothesis is that BCL-3 directly regulates class switch recombination and somatic hypermutation because these genetic alterations have a close correlation with the transcriptional activation of the Ig gene itself and the intronic switch region (27Honjo T. Kinoshita K. Muramatsu M. Annu. Rev. Immunol. 2002; 20: 165-196Crossref PubMed Scopus (501) Google Scholar, 28Martin A. Scharff M.D. Nat. Rev. Immunol. 2002; 2: 605-614Crossref PubMed Scopus (665) Google Scholar, 29Manis J.P. Tian M. Alt F.W. Trends Immunol. 2002; 23: 31-39Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In this study we identified B3BP as a protein that specifically interacts with BCL-3. It was shown that B3BP also interacts with histone acetyltransferase p300/CBP and that the ATP-binding site of B3BP is important for the association with BCL-3 or p300/CBP. Biochemical analysis revealed that B3BP has polynucleotide kinase activity to transfer a phosphate group to the 5′ end of DNA and RNA substrates. Moreover, a small MutS-related (Smr) 1The abbreviations used are: Smr, small MutS-related; HA, hemagglutinin; ORF, open reading frame; B3BP, BCL-3-binding protein; CNP, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; PNK, polynucleotide kinase; GST, glutathione S-transferase; ytRNA, yeast transfer RNA; CBP, cAMP-responsive element-binding protein; CBB, Coomassie Brilliant Blue; 8-azido-ATP, 8-azidoadenosine-5′-triphosphate; MES, 2-[N-morpholino]ethanesulfonic acid.1The abbreviations used are: Smr, small MutS-related; HA, hemagglutinin; ORF, open reading frame; B3BP, BCL-3-binding protein; CNP, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; PNK, polynucleotide kinase; GST, glutathione S-transferase; ytRNA, yeast transfer RNA; CBP, cAMP-responsive element-binding protein; CBB, Coomassie Brilliant Blue; 8-azido-ATP, 8-azidoadenosine-5′-triphosphate; MES, 2-[N-morpholino]ethanesulfonic acid. domain found in the C-terminal region of B3BP exhibits nicking endonuclease activity, which is postulated to have a role in mismatch repair or genetic recombination (30Malik H.S. Henikoff S. Trends Biochem. Sci. 2000; 25: 414-418Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 31Moreira D. Philippe H. Trends Biochem. Sci. 1999; 24: 298-300Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). These findings suggest that B3BP plays a role connecting transcriptional activation and genetic recombination of the Ig gene. Yeast Two-hybrid Screening—A fragment of mouse BCL-3 cDNA encoding amino acids 16–440 cloned into the LexA DNA-binding domain vector pBTM116 and a human spleen cDNA library cloned into the GAL4 activation domain vector pGAD10 (Clontech) were sequentially used to transform L40 yeast cells according to the protocol described on the Yeast Transformation Home Page ( www.umanitoba.ca/faculties/medicine/units/biochem/gietz/Trafo.html ). Transformants were plated on selection medium (lacking tryptophan, leucine, and histidine) containing 10 mm 3-amino-1,2,4-triazole. After incubation for 7 days at 25 °C, the clones that allowed growth were identified and confirmed to express β-galactosidase. The plasmids were recovered according to Matchmaker protocols (Clontech) and retransformed into yeast cells containing either pBTM116-BCL-3 or pBTM116 empty vector. cDNA inserts from plasmids that allowed the yeast cells containing pBTM116-BCL-3 to grow on selection medium were identified and further characterized by DNA sequencing. cDNA Cloning and Plasmid Construction—To obtain a full-length B3BP cDNA, a human T cell cDNA library carried in λZAP Express (Stratagene) was screened under stringent conditions using the cDNA of a ∼4.7-kb insert isolated in the two-hybrid screen as a probe. The cDNA inserts from positive phage clones were excised in vivo to generate subclones in the pBK-CMV phagemid and confirmed by sequencing. The ORF containing full-length B3BP cDNA was assembled on a mammalian expression vector, pEF-BOS (32Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1489) Google Scholar). To express GST fusion proteins in Escherichia coli JM109, DNA fragments encoding amino acids 2–631, 1171–1770, 394–630, and 1688–1770 of B3BP were cloned into pGEX-4T (Amersham Biosciences) to construct pGEX-B3BP(N), pGEX-B3BP(C), pGEX-B3BP(M), and pGEX-B3BP(Smr), respectively. A cDNA encoding full-length mouse BCL-3 was cloned into pGEX-4T to express GST-BCL-3 fusion protein. cDNA fragments encoding B3BP, BCL-3, BCL-3 derivatives, and NFKB1 were subcloned into pCS2+ (33Rupp R.A. Snider L. Weintraub H. Genes Dev. 1994; 8: 1311-1323Crossref PubMed Scopus (564) Google Scholar) to transcribe and translate in rabbit reticulocyte lysate (TnT SP6 Quick System; Promega). HA-tagged expression vectors were constructed by inserting the DNA fragment encoding MGYPYDVPDYASLGG for pEF-HA-B3BP and pEF-HA-BCL-3 in the N-terminal end of B3BP and BCL-3, respectively. HA-tagged p300 and FLAG-tagged CBP constructs were described previously (34Suhara W. Yoneyama M. Kitabayashi I. Fujita T. J. Biol. Chem. 2002; 277: 22304-22313Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). FLAG-tagged constructs were obtained by inserting the DNA fragment encoding GPGDYKDDDKGDYKDDDK for pEF-B3BP-FLAG and pEF-BCL-3-FLAG in the C-terminal end of B3BP and BCL-3, respectively. To construct expression vectors encoding B3BP derivatives, B3BP(K/A) and B3BP(K/R), site-directed mutagenesis of the Lys453 residue within the ATP-binding site of B3BP was conducted using QuikChange™ (Stratagene) according to the manufacturer's instructions. The authenticity of the substitutions and the absence of any undesired mutations were confirmed by sequence analysis. Antibodies—The monoclonal antibodies against the HA epitope (12CA5; Roche Applied Science), FLAG epitope (M2; Sigma-Aldrich), and p300/CBP (mixed monoclonal antibodies; Upstate Biotechnology, Inc.) were obtained commercially. The monoclonal antibody against NFKB1 was described previously (35Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar). Recombinant Proteins and in Vitro Binding Assay—All of the pGEX-based bacterial expression vectors were transformed into E. coli strain JM109, and GST fusion proteins were expressed and purified using glutathione-Sepharose resin according to the manufacturer's directions (Amersham Biosciences). For the in vitro biochemical analysis, GST, GST-B3BP(M), and GST-B3BP(Smr) were eluted from the column with buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm dithiothreitol, and 20 mm glutathione) and dialyzed against phosphate-buffered saline. Subsequently, GST-B3BP(Smr) was treated with thrombin protease, and the Smr domain was further purified by ion-exchange chromatography on a Mono S column (Amersham Biosciences). For the GST-based interaction assay, GST fusion proteins attached to glutathione matrix beads were incubated with rabbit reticulocyte lysate containing 35S-radiolabeled protein in Nonidet P-40 binding buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1% (v/v) Nonidet P-40, and 10% (v/v) glycerol) for 2 h at 4 °C. The beads were subsequently washed five times with Nonidet P-40 binding buffer, and the bound proteins were fractionated by SDS-PAGE and visualized by autoradiography or Coomassie Brilliant Blue (CBB) staining. Reticulocyte lysate used in Fig. 5B was ATP-depleted by passing through the Sephadex G-50 (Amersham Biosciences) column. Cell Culture, DNA Transfection, Immunoprecipitation, and Western Blotting—293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected by the calcium phosphate method. 48 h after transfection, 293T cells (∼2 × 106 cells/6-cm dish) were harvested, washed with phosphate-buffered saline, and suspended in 400 μl of the extraction buffer (20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 5 mm EDTA, 0.5% (v/v) Triton X-100, 100 μg of leupeptin/ml, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 10% (v/v) glycerol, and 10 mm benzamidine). Subsequently, the suspensions were allowed to stand on ice for 30 min and clarified by centrifugation (356,000 × g, 10 min), and the resulting supernatant was subjected to immunoblot analysis and immunoprecipitation. For the FLAG-based interaction assay, typically 100 μl of cell lysate prepared as described above was incubated with 10 μl of anti-FLAG M2-agarose (Sigma-Aldrich) in the extraction buffer for 2 h at 4 °C. Immunoprecipitates were washed five times with the extraction buffer, resuspended in 1× Laemmli's sample buffer, and subjected to SDS-PAGE. The proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore Corp.) and incubated with anti-HA (12CA5) and anti-FLAG (M2) monoclonal antibodies. Subsequently they were visualized with appropriate secondary antibodies conjugated with horseradish peroxidase and an ECL+plus Western blotting detection system (Amersham Biosciences). Assay for ATP Binding and Hydrolyzing Activity—B3BP-FLAG and its derivatives were expressed in 293T cells and purified using anti-FLAG M2-agarose resin as described above. For the ATP binding assay, proteins attached to the beads were suspended in buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm MgCl2, and 0.1% (v/v) Tween 20) and subjected to UV irradiation in the presence of α-32P-labeled 8-azidoadenosine-5′-triphosphate (8-azido-ATP). After the washing out of the uncross-linked 8-azido-ATP, the proteins were separated by SDS-PAGE and visualized by autoradiography or CBB staining. For the ATP hydrolyzing assay, the proteins were cross-linked with γ-32P-labeled 8-azido-ATP and incubated in the presence or absence of 10 μg of yeast transfer RNA/ml for 60 min at 30 °C in the buffer described above. After a wash, the proteins were separated by SDS-PAGE, visualized by autoradiography, and quantified using a Bio Image Analyzer BAS-2500 (FUJIFILM). The amount of protein subjected to the assay was confirmed by CBB staining. Assay for Polynucleotide Kinase Activity—Recombinant proteins, GST and GST-B3BP(M), or FLAG-tagged B3BP and its derivatives expressed in 293T cells and absorbed onto anti-FLAG resin were incubated in buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, and 5 mm MgCl2) at 37 °C for 1 h in the presence of γ-32P-labeled ATP with the following substrates: single-stranded DNA, 3′OH-GGAATTCAGAATGTTTTAGCCCTCCATGGGCTGCATGTGG-5′OH; double-stranded DNA, 40 bp annealed with 3′OH-GGAATTCAGAATGTTTTAGCCCTCCATGGGCTGCATGTGG-5′OH and 3′OH-GCTTCATCCACATGCAGCCCAGCGAGGTCTAAAACATTCTG-5′OH, yeast transfer RNA (Roche Applied Science). The samples were separated on a denaturing 10% polyacrylamide gel and visualized by autoradiography. The proteins subjected to the assay were run on a NuPAGE 4–12% Bis-Tris gel with SDS-containing MES buffer (Invitrogen) and visualized by CBB staining. Assay for Nicking Endonuclease Activity—Recombinant proteins were incubated in buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, and 50 μg of bovine serum albumin/ml) at 37 °C for 2 h with a supercoiled circular DNA (pEF-BOS) as a substrate. The samples were separated on 1% agarose gel and visualized by ethidium bromide staining. The proteins subjected to the assay were run on a NuPAGE 4–12% Bis-Tris gel with SDS-containing MES buffer (Invitrogen) and visualized by CBB staining. Identification of B3BP as a Protein Interacting with BCL-3— We sought to identify proteins other than NFKB1 and NFKB2 that interact specifically with BCL-3. We set up a yeast two-hybrid system with full-length BCL-3 as bait and screened a GAL4 activation domain-tagged cDNA library derived from the human spleen. Although full-length BCL-3 activates transcription substantially in yeast when tethered to DNA via the LexA DNA-binding domain, a competitive inhibitor of His3p, 3-amino-1,2,4-triazole, completely abrogated its intrinsic activity, and screening worked properly under these conditions. From a screen of ∼6 × 106 colonies, 38 clones grew on the selective medium and showed β-galactosidase activity. One clone had a 4.7-kb insert, and a protein of corresponding molecular mass (∼200 kDa) was detected by anti-GAL4 activation domain antibody in the yeast lysate (data not shown). Subsequent sequence analysis revealed that it contains a large in-frame ORF, and a data base search indicated that a portion of its cDNA has been registered as KIAA1413 with unknown function. We obtained full-length cDNA by screening a human T cell cDNA library using the 4.7-kb insert as a probe. The largest clone was isolated and sequenced. It contained a 6626-bp cDNA insert and extended 132 bp 5′ of a Kozak consensus sequence for the predicted start of translation. The predicted polypeptide specified by the ORF comprised 1770 amino acids, which was calculated to be ∼200 kDa. Recently, a 374-amino acid fragment near the C-terminal region of this predicted polypeptide was identified by yeast two-hybrid screening and reported as N4BP2 (Nedd4 ubiquitin ligase-binding partner 2) (36Murillas R. Simms K.S. Hatakeyama S. Weissman A.M. Kuehn M.R. J. Biol. Chem. 2002; 277: 2897-2907Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and its full-length protein was referred to as flN4BP2. It was demonstrated that the corresponding region specifically associates with Nedd4 in vitro and in vivo and was ubiquitinated by Nedd4 in vitro. There has been no biochemical characterization of flN4BP2 so far; therefore we refer to it as BCL-3-binding protein in this paper. Salient features of B3BP include a consensus nucleotide-binding site, the Walker A motif, at residues 447–454, GLPGSGKS. Homology searches using the BLAST algorithm indicate that the nucleotide binding motif and its neighboring sequence most resemble those of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) of mammals and polynucleotide kinase (PNK) of bacteriophage T4 (PNK domain; Fig. 1, A and B), both of which possess 5′-polynucleotide kinase activity. The other motif shown by the analysis is the Smr domain found in the C-terminal region of B3BP (Fig. 1, A and C). The Smr domain has been described as a highly conserved sequence in the C-terminal region of the bacterial MutS2 family and phylogenetically speculated to be involved in DNA mismatch repair and meiotic chromosome crossing-over (30Malik H.S. Henikoff S. Trends Biochem. Sci. 2000; 25: 414-418Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 31Moreira D. Philippe H. Trends Biochem. Sci. 1999; 24: 298-300Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We also isolated a mouse homolog of B3BP cDNA that encodes a protein ∼70% identical to the human protein (data not shown). Notably, the N- and C-terminal regions of 300 amino acids containing the PNK and Smr domains, respectively, are more than 88% identical, suggesting functional conservation of these domains. Furthermore, in a survey of the Drosophila melanogaster genome data base, we found a putative ORF, LD21293, that encodes a polypeptide containing both the PNK and Smr domains at its N and C termini, respectively. However, the region between these domains exhibits no significant similarity. There is no ORF that encodes a single polypeptide containing these domains in the C. elegans or Saccharomyces cerevisiae genome, although stand-alone ORFs that encode the Smr domain are found in many lineages of eukaryotic and prokaryotic genomes (30Malik H.S. Henikoff S. Trends Biochem. Sci. 2000; 25: 414-418Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 31Moreira D. Philippe H. Trends Biochem. Sci. 1999; 24: 298-300Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). B3BP Interacts with BCL-3 in Vitro—The interaction between B3BP and BCL-3 was confirmed in vitro. GST fusion proteins containing the N-terminal (amino acids 2–631; GST-B3BP(N)) or C-terminal (amino acids 1171–1770; GST-B3BP(C)) regions of B3BP were expressed in E. coli and affinity-purified on glutathione-Sepharose beads. These proteins were then incubated with 35S-labeled BCL-3 translated in rabbit reticulocyte lysate. After extensive washing, the bound proteins were separated on an SDS-polyacrylamide gel and visualized by autoradiography. As shown in Fig. 2A, BCL-3 bound to the N-terminal portion but not the C-terminal portion of B3BP (lanes 5 and 6). In a converse experiment, GST-BCL-3 fusion protein bound to full-length B3BP (lane 7). The coprecipitation of BCL-3 and GST-BCL-3 is due to homophilic interaction (37Wulczyn F.G. Naumann M. Scheidereit C. Nature. 1992; 358: 597-599Crossref PubMed Scopus (181) Google Scholar, 38Michel F. Soler-Lopez M. Petosa C. Cramer P. Siebenlist U. Muller C.W. EMBO J. 2001; 20: 6180-6190Crossref PubMed Scopus (58) Google Scholar). We used luciferase, which is unrelated to BCL-3 or B3BP, as a negative control and found that it bound neither to GST nor to GST fusion proteins. It has been shown that the ankyrin repeat domain of BCL-3 is important for the interaction of the homodimer of the NFKB1 p50 subunit (38Michel F. Soler-Lopez M. Petosa C. Cramer P. Siebenlist U. Muller C.W. EMBO J. 2001; 20: 6180-6190Crossref PubMed Scopus (58) Google Scholar). Therefore, we next examined the involvement of the ankyrin repeat domain in the association with B3BP (Fig. 2B). Wild type BCL-3 and the C-terminal deletion mutant, BCL-3(ΔC), which lacks the C-terminal portion (amino acids 353–437) but retains the entire ankyrin repeat domain, specifically associated with GST-B3BP(N) (lanes 5–8). However, another mutant BCL-3(Δank), which lacks a part of the ankyrin repeat (amino acids 260–339) did not bind to GST-B3BP(N) (lanes 9 and 10), indicating the importance of the ankyrin repeat domain. BCL-3(Δank) failed to interact with the NFKB1 p50 subunit as well (data not shown). Although the ankyrin repeat domains of BCL-3 and NFKB1 and NFKB2 are homologous, no significant association was detected between NFKB1 and GST-B3BP(N) (lanes 11 and 12). B3BP Also Interacts with Histone Acetyl Transferase p300/CB" @default.
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• W2058258296 title "Identification and Characterization of BCL-3-binding Protein" @default.
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